Endothelium-derived nitric oxide (EDNO) is a potent endogenous vasodilator and is a key mediator of physiological demand for increased blood flow. In addition, EDNO inhibits key processes that promote atherosclerosis, such as vascular smooth muscle proliferation, platelet adhesion and aggregation, and leukocyte-endothelial cell adhesion. Thus, reduced EDNO activity is a common cause of vascular dysfunction and may be instrumental in the development and progression of atherosclerosis. EDNO is derived from L-arginine by the constitutive endothelial isozyme of nitric oxide synthase (NOS). Recently, asymmetric dimethyl arginine (ADMA) was identified as an endogenous competitive inhibitor of endothelial NOS. Blood ADMA is chronically elevated up to 10-fold in patients with vascular disease and at least 2-fold in healthy adults with high serum cholesterol who are considered at risk for atherosclerosis. Serum ADMA correlates better than cholesterol with other indicators of vascular dysfunction such as reduced NO activity, impaired vasodilation, and increased monocyte adhesion to the endothelium. Thus, ADMA may be more reliable than serum cholesterol as a risk indicator for vascular disease. ADMA is currently assayed by a cumbersome, time-consuming, and expensive procedure involving extraction, chemical derivatization, isolation by reverse-phase HPLC, and detection by fluorescence. Such procedures may preclude routine monitoring of this important risk factor. The investigators propose a novel homogeneous enzymatic immunoassay by which blood ADMA levels could be measured rapidly and accurately with inexpensive materials and equipment. The assay will be based on the activity of beta-galactosidase (beta-gal), which has been engineered to be sensitive to ADMA. The engineered enzyme will display a peptide analog of ADMA that inhibits enzyme activity when, and only when, it is bound by an anti-ADMA antibody. In the presence of free ADMA, the antibody inhibitor is displaced from the enzyme, and activity increases proportionately. This assay will expedite basic and clinical studies of the role of ADMA in vascular disorders, and investigators believe the assay will ultimately become an important tool for the clinician in the assessment of cardiovascular risk as well as in the analysis of vascular response to treatment. In Phase I, investigators will develop a fully functional prototype of the assay. In Phase 2, the ADMA immunoassay will be refined and tested clinically and will be used to further studies of the role of ADMA in the regulation of vascular tone and in the etiology of vascular disease. The ultimate goal of Phase 2 will be to obtain approval to market the assay for cardiovascular disease risk assessment. There are three specific aims to Phase I. First, to obtain and characterize a panel of monoclonal antibodies against haptenized ADMA. Second, to obtain a panel of peptide epitope analogs of ADMA by panning a phage display library of random peptides against immobilized anti-ADMA monoclonal antibodies. Third, to express peptide epitope analogs as beta-galactosidase fusions and test for antibody-dependent inhibition of beta-galactosidase and reactivation by free ADMA. Investigators present comprehensive and detailed methodology on how to accomplish these three Phase I goals. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE